Organ transplant rejection syndrome is well documented. In this situation, organs transplanted to a recipient from a donor are bound by antibodies circulating in the blood of the recipient, that is, anti-HLA antibodies. Organ rejection leads to loss and destruction of the transplanted organ, and acute medical and pathological problems for the recipient.
Two assays have been developed in the art to ascertain the presence of donor-specific antibodies in proposed transplant recipients. In one assay, a compliment-dependent microcytotoxicity assay (CDL) using peripheral blood lymphocytes (PBL) of the donors is employed. This technique is well documented, and, in the case of renal transplants, employs a modified Amos technique. Polymorphs and macrophages are separated off, and mononuclear cells may be further purified with T-B Kwik (one lambda, California). This prior art assay method calls for incubation of cells with sera (from the proposed recipient) at 25.degree. C. for 30 minutes, the cells being washed twice and then incubated with goat anti-human kappa for 2 minutes before adding rapid compliment at 25.degree. C. for 1 hour. Cell death is determined by eosin staining followed by formalin fixation. As an alternative assay, indirect immunofluorescence (IF) assays have been previously developed. The inventor, working together with others, developed this technique (IF) to increase sensitivity and specificity of the assay. Lobo et al., Transplantation 23:16-21 (1977). This assay was particularly characterized by pre-digestion of the donor lymphocytes with pronase to remove Fcy R receptors that bind to the Fc region of normal or irrelevant IgG. This is a requirement to avoid false negative assays, as it is impossible to distinguish binding of normal or irrelevant IgG from anti-HLA binding. See, Lobo et al., J. Immunol. 117:939-942 (1976) and Lobo et al., Hum. Immunol. 1:55-60 (1981).
IF assays proved to be difficult in a clinical environment, however, in part due to the requirements for ultraviolet microspy (X800 magnification) which renders the test highly subjective, and cumbersome.
In 1983, an improved method for detecting anti-HLA antibody binding to donor lymphocytes was developed, employing flow cytometry instead of microscopy to quantitate antibody binding. Garovoy et al., Transplant Proc. 15:1939-1945 (1983). This assay, or flow cytometric crossmatch (FCXM) has received broad attention in the art. In FCXM, using normal control sera and donor sera, a fluorescence histogram is obtained. A curser is set at the point on the histogram where most or all of the cells to the left of the curser are considered to be fluorescent-negative. A serum that increases fluorescence to the right of the curser is considered positive. Given the presence of receptors that bind to normal or irrelevant IgG in normal patient sera, however, it has been found difficult to differentiate IgG binding, and to identify sera that are truly negative for anti-HLA antibodies. Thus, the FCXM assay developed lacks specificity.
In an attempt to answer this lack of specificity, a two-color system was developed, where T or B or lymphocytes were identified with phycoerythrin-labeled anti-T or -B murine monoclonal antibodies, to avoid binding results of irrelevant or normal IgG on the non-B, non-T-lymphocyte subset. This technique continues to experience "false-positive" problems. Ogura et al., Transplantation 56:294-298 (1993) and Scornik et al., Transplantation 57:621-625 (1994).
Thus, although superior to CDL in identifying anti-HLA antibodies in the sera of proposed organ recipients, FCXM lacks specificity. IF, on the other hand, is cumbersome and subjective.
Retransplant recipients pose a special problem for detection of low titer (or weak) HLA class I antibodies that bind only to B cells. These recipients typically have high T cell panel reactive antibodies (PRA).
Thus, it remains an object of those of ordinary skill in the art to develop an assay that is objective, sensitive and specific, that will allow the reliable detection of anti-HLA antibodies in proposed transplant recipient patients, and avoid problems of both false positive and false negative results.